Dynamin-dependent transferrin receptor recycling by endosome-derived clathrin-coated vesicles

Abstract

Previously we described clathrin-coated buds on tubular early endosomes that are distinct from those at the plasma membrane and the trans-Golgi network. Here we show that these clathrin-coated buds, like plasma membrane clathrin-coated pits, contain endogenous dynamin-2. To study the itinerary that is served by endosome-derived clathrin-coated vesicles, we used cells that overexpressed a temperature-sensitive mutant of dynamin-1 (dynamin-1(G273D)) or, as a control, dynamin-1 wild type. In dynamin-1(G273D)-expressing cells, 29-36% of endocytosed transferrin failed to recycle at the nonpermissive temperature and remained associated with tubular recycling endosomes. Sorting of endocytosed transferrin from fluid-phase endocytosed markers in early endosome antigen 1-labeled sorting endosomes was not inhibited. Dynamin-1(G273D) associated with accumulated clathrin-coated buds on extended tubular recycling endosomes. Brefeldin A interfered with the assembly of clathrin coats on endosomes and reduced the extent of transferrin recycling in control cells but did not further affect recycling by dynamin-1(G273D)-expressing cells. Together, these data indicate that the pathway from recycling endosomes to the plasma membrane is mediated, at least in part, by endosome-derived clathrin-coated vesicles in a dynamin-dependent manner.

Figure 1

Clathrin-coated buds on endosomes contain endogenous dynamin. Nontransfected HeLa cells were incubated in the presence of Tf/HRP for 60 min at 37°C, and processed for whole mount immunoelectron microscopy. Samples were immuno-double–labeled for clathrin (cla, 10-nm gold) and endogenous dynamin-2 (dyn2, 5-nm gold) using polyclonal anti-clathrin and monoclonal HUDY-1, respectively. (A–C) Tubular early endosomes varied in length (compare A with B), but all were decorated with buds that labeled for both clathrin and dynamin-2 (for examples, see arrowheads). For quantification, see Figure 9A. (B) A large electron-lucent dynamin/clathrin-coated pit at the plasma membrane (arrow) is clearly different from DAB-contrasted 60-nm buds on endosomes (arrowheads). (C) Occasionally, tubules and buds (arrowhead) seem to associate with early endosomal vacuoles (asterisk). Bar, 200 nm.

Figure 2

Expression of dyn^wt and dyn^ts and the effects on TfR internalization. (A) Cells were cultured for 6 d at 32°C in the presence (+) or absence (−) of tetracycline for the last 1–6 d. After extensive washing and replacement of tetracycline-containing medium by tetracycline-lacking medium, medium was either left on the cells for 6 d (− refresh medium) or refreshed daily (+ refresh medium). Western blots of cell lysates were probed with anti-HA antibodies to demonstrate HA-tagged dyn^wt or dyn^ts and with anti-tubulin antibodies as a control to demonstrate equal amounts of cell lysate. Maximal expression levels of dyn^wt and dyn^ts were observed 3–4 d after removal of tetracycline and daily replacement of the culture medium. (B) dyn^wt- or dyn^ts-expressing cells were preincubated for 30 min at either 25 or 38°C and directly thereafter incubated in the presence of ¹²⁵I-Tf at the same temperature for the indicated times. Internalized ¹²⁵I-Tf was expressed as a percentage of total cell-associated ¹²⁵I-Tf (n = 2 ± SD). The results shown are representative of three independent experiments. In comparison with dyn^wt cells (○), uptake of ¹²⁵I-Tf by dyn^ts cells (□) was strongly reduced at 38°C but not at 25°C.

Figure 3

Inhibited TfR recycling by dyn^ts. (A and B) dyn^wt (○) and dyn^ts (□)-expressing cells were loaded with ¹²⁵I-Tf for 1 h at either 25°C (A) or 38°C (B). Cell surface-associated ¹²⁵I-Tf was removed at 0°C, after which recycling of endocytosed ¹²⁵I-Tf was measured during continued incubations at 25°C (A) or 38°C (B). Intracellular ¹²⁵I-Tf is plotted as a percentage of total endocytosed ¹²⁵I-Tf (n = 2, mean ± SD). The experiments shown are representative of 13 independent experiments, each with duplicate samples. For kinetic analysis, see Table 1. (C and D) Sorting endosomes of dyn^wt (○) and dyn^ts (□)-expressing cells were loaded with ¹²⁵I-Tf for 1 h at 16°C. Cell surface-associated ¹²⁵I-Tf was removed at 0°C, and recycling of endocytosed ¹²⁵I-Tf was measured at either 25°C (C) or 38°C (D). Intracellular ¹²⁵I-Tf is plotted as a percentage of total endocytosed ¹²⁵I-Tf (n = 2, mean ± SD). The experiments shown are representative of two to five independent experiments, each with duplicate samples. For the kinetic analysis, see Table 1. (E) dyn^wt (○) and dyn^ts (□)-expressing cells were loaded with ¹²⁵I-Tf-SS-biotin for 1 h at 38°C. Cell surface-associated ¹²⁵I-Tf-SS-biotin was removed at 0°C, after which recycling of endocytosed ¹²⁵I-Tf-SS-biotin was allowed at 38°C for the times indicated. The biotin moiety was then removed selectively from surface-exposed ¹²⁵I-Tf-SS-biotin by exposing the cells to MESNA at 0°C. Intracellular ¹²⁵I-Tf-SS-biotin was then collected from cell lysates and plotted as a percentage of total internalized ¹²⁵I-Tf-SS-biotin (mean ± SD from two independent experiments). For the kinetic analysis, see Table 1.

Figure 4

Inhibition of Tf recycling by dyn^ts is not due to defective endosome acidification. (A) dyn^wt (circles) and dyn^ts (squares)-expressing cells were loaded with ¹²⁵I-Tf for 1 h at 38°C. Cell surface-associated ¹²⁵I-Tf was removed at 0°C by the acidic-neutral wash procedure using either MES buffer (open symbols) or acetate buffer (closed symbols). The cells were then reincubated at 38°C, and the release of ¹²⁵I-Tf was measured. Intracellular ¹²⁵I-Tf is plotted as a percentage of total endocytosed ¹²⁵I-Tf (n = 2, mean ± SD). The experiments shown are representative of three independent experiments, each with duplicate samples. The acetate treatment assured the release of Fe³⁺ from intracellular ¹²⁵I-Tf. dyn^ts inhibited the release of ¹²⁵I-Tf irrespective of acetate or MES treatment. Lack of endosome acidification thus can not explain the results. (B) As a positive control for this procedure, dyn^wt-expressing cells were loaded for 1 h at 38°C with ¹²⁵I-Tf in the absence (○) or presence (▵ and □) of 100 nM of concanamycin A, a vacuolar proton pump inhibitor. Cell surface-associated ¹²⁵I-Tf was removed at 0°C by the acid-neutral wash procedure using either MES buffer (□) or acetate buffer (○ and▵). Cells were then reincubated at 38°C in the absence (○) or presence (▵ and □) of 100 nM concanamycin A and release of ¹²⁵I-Tf was measured. Intracellular ¹²⁵I-Tf is plotted as a percentage of total internalized ¹²⁵I-Tf. The results shown (n = 2, mean ± SD) are representative of five independent experiments. The acetate treatment restored ¹²⁵I-Tf recycling by concanamycin A-treated cells, illustrating the effectiveness of this procedure.

Figure 5

dyn^ts cells redistribute intracellular TfR. dyn^wt (A–C) and dyn^ts (D–F)-expressing cells were incubated for 30 min at 38°C, fixed, and immuno-double–labeled for dynamin-1 (green) and TfR (red). Integrated views of entire cells were obtained by superimposition of 25 sequential 0.3-μm optical sections. Note that in dyn^ts cells dynamin-1 accumulated at the edge of the cell and in large aggregates. TfR accumulated at the plasma membrane as well as in tubular endosomes in the perinuclear area. The latter structure apparently also accumulated dyn^ts. Bar, 10 μm.

Figure 6

dyn^ts cells accumulate endocytosed Tf in a perinuclear compartment. dyn^wt (A–F) and dyn^ts (G–L)-expressing cells were pulse-labeled with both TaR-Tf and F-dextran for 1 h at 38°C. Extracellular and cell surface-associated labels were removed at 0°C by the acid-neutral wash procedure. The cells were then either fixed immediately (A–C, G–I) or chased for 1 h at 38°C before fixation (D-F, J-L). dyn^wt cells released almost all TaR-Tf during the chase, whereas dyn^ts cells retained significant amounts of TaR-Tf in F-dextran lacking endosomes that localize both at the perinuclear area and at the periphery of the cell. Bar, 10 μm.

Figure 7

dyn^ts cells accumulate Tf in recycling endosomes. dyn^ts-expressing cells were loaded with TaR-Tf for 1 h at 38°C. Cell surface-associated TaR-Tf was removed at 0°C by the acid-neutral wash procedure and the cells were chased for 1 h at 38°C, fixed, permeabilized, and immunolabeled for either TGN-46 or EEA1. TaR-Tf was retained in peripheral and perinuclear endosomes that are distinct from the TGN (TGN-46) and sorting endosomes (EEA1). Bar, 10 μm.

Figure 8

dyn^ts localizes on endosomal clathrin-coated buds. dyn^wt (A and C) and dyn^ts (B and D) cells were incubated for 1 h at 25°C with Tf/HRP to allow loading of the entire recycling pathway with HRP activity. The cells were then incubated for 5 min at 38°C to initiate the dominant-negative effect of dyn^ts. The cells were processed for whole mount immunoelectron microscopy and labeled for dynamin-1 (dyn^wt or dyn^ts, 10-nm gold) and clathrin (cla, 5-nm gold; A and B) or dynamin-1 (10-nm gold) and TfR (5-nm gold; C and D). (A) Clathrin-coated buds on tubular endosomes in dyn^wt cells are largely devoid of dyn^wt. (B) Clathrin-coated buds on extended tubular endosomal networks in dyn^ts cells are mostly labeled for dyn^ts (for examples, see arrowheads). For quantification, see Figure 9. (C) TfR-labeled tubular endosomes in dyn^wt cells contain little, if any, dyn^wt. (D) TfR-labeled endosomes in dyn^ts cells contain many dyn^ts-labeled buds (arrowheads). Limited labeling for TfR on dyn^ts-containing buds was probably due to stearic hindrance. Bars, 200 nm.

Figure 9

Dynamin and clathrin codistribute on endosomal buds. (A) Double-labeled whole mount immunoelectron microscopic preparations of nontransfected HeLa cells (see Figure 1) were analyzed for the presence of endogenous dynamin-2 and clathrin on endosomal buds. Of 300 immunolabeled DAB-positive buds that were counted, ∼93% contained both endogenous dynamin-2 and clathrin. (B) Double-labeled whole mount immunoelectron microscopic preparations of dyn^ts cells (see Figure 8B) were analyzed for the presence of dyn^ts and clathrin on endosomal buds. Of 700 labeled DAB-positive buds that were counted, ∼90% contained both dyn^ts and clathrin.

Figure 10

Interference of TfR recycling by BFA. (A and B) dyn^wt (circles) and dyn^ts (squares)-expressing cells were loaded with ¹²⁵I-Tf for 1 h at either 25°C (A) or 38°C (B). Cell surface-associated ¹²⁵I-Tf was removed at 0°C, and the cells were reincubated at 25°C (A) or 38°C (B) in the absence (open symbols) or presence (closed symbols) of 10 μg/ml BFA, and the release of ¹²⁵I-Tf was measured. Intracellular ¹²⁵I-Tf is plotted as a percentage of total endocytosed ¹²⁵I-Tf (n = 2, mean ± SD). The experiments shown are representative of two independent experiments each with duplicate samples. For the kinetic analysis see Table 1. At 25°C BFA interfered with ¹²⁵I-Tf recycling by both dyn^wt and dyn^ts cells. In contrast, at 38°C BFA had no additive effect on ¹²⁵I-Tf recycling by dyn^ts cells.